Why Do Proteins Get Stuck and Fail to Move Down in Western Blot?
When performing Western blot, if proteins get stuck and fail to migrate, it could be due to several reasons:
1. Electrophoresis Conditions
(1) Low Voltage: If the voltage is too low, the protein migration speed will be slow, making it seem like the proteins are stuck.
(2) Insufficient Running Time: If the electrophoresis time is too short, proteins may not have enough time to travel through the gel.
2. Gel Issues
(1) Inappropriate Pore Size: If the gel’s pore size is too small, large molecular weight proteins may not be able to pass through.
(2) Too High Gel Concentration: High-concentration gels are better suited for separating small proteins, which may make it difficult for larger proteins to migrate.
3. Sample Issues
(1) High Sample Concentration: Excessive protein concentration can cause proteins to aggregate in the gel wells.
(2) Insufficient Denaturation or Reduction: If the sample is not fully denatured or reduced, proteins may remain in aggregated or secondary structures, hindering their migration.
4. Buffer Issues
Inappropriate Buffer Composition: The pH or ionic strength of the buffer could also affect protein migration.
Solutions
1. Adjust Electrophoresis Conditions
Increase voltage or extend running time.
2. Choose Appropriate Gel
Use lower concentration or larger pore size gels to facilitate large protein migration.
3. Optimize Sample Preparation
Ensure samples are fully denatured and reduced, and avoid high concentrations.
4. Adjust Buffer Composition
Use a buffer with the proper composition.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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